Matriptase is a cell-surface anchored serine protease that was first identified in human breast cancer cell lines and has subsequently been implicated in many aspects of breast cancer pathology. Matriptase is up-regulated in human breast carcinoma cancer cells, and its increased expression has been shown to correlate with poor patient outcome. Presently, it is unknown if matriptase plays a causal role in breast carcinogenesis or contributes primarily to secondary events of cancer growth and progression, and its potential as a therapeutic target remains untested. Perinatal lethality in matriptase loss-of-function mice has thus far precluded analysis of the effect of matriptase ablation in the mammary gland; however our proposal presents novel techniques to bypass this limitation. In our proposed study both functional and mechanistic characterization of matriptase's role in breast cancer will be performed using parallel and complimentary in vitro and in vivo loss-of-function techniques. Our previous research has identified the GPI-anchored serine protease prostastin as a physiological substrate and downstream effector of matriptase proteolytic activity, and we will focus on this proteolytic axis for its potential contribution to breast cance biology. We believe there to be a significant correlation between these two proteases and cancer progression, as we have demonstrated that both matriptase and prostasin play critical roles in cell to cell adhesions in vivo, and changes in levels of either protein can perturb these adhesions via disruption of epithelial tight junction formation and integrity. The hypothesis to be tested is that matriptase exerts critical functions through activation of the prostasin zymogen and that the matriptase/prostasin proteolytic pathway is critical for breast oncogenesis, specifically through the loss of cell-cell adhesions, and thereby the promotion of carcinogenesis in vivo. To test this hypothesis, we formulated two specific aims. In the first aim the significance of the matriptase/prostasin proteolytic axis in breast cancer initiation and progression will be determined using both matriptase and prostasin loss-of-function novel genetic mouse models. In the second aim the role of prostasin in cell-cell adhesion in breast cancer will be studied with emphasis on its role in tight junction function and integrity. The combination of state-of-the art mouse genetics with 2D and 3D cell culture based assays encompasses an innovative strategy for studying human cancerous disease, and may offer new avenues for diagnosis and therapy of breast cancer. PUBLIC HEALTH RELEVANCE: Extracellular proteases are the subject of intensive efforts by the pharmaceutical industry to develop new treatment strategies for human diseases, including breast cancer. We propose to study and validate a protease pathway, the matriptase-prostasin proteolytic pathway, that is active in the tumor microenvironment and that may mediate the progression from normal breast epithelium to malignant and metastatic lesions. We anticipate that our studies will identify a novel target which can be used to develop strategies to abrogate breast cancer progression.